This invention is related to a sample stage for use on an ion mass spectrometry system. In particular, this invention is directed to a cryogenic sample stage which allows the study of the true distribution of ions in biological systems using an ion microscope.
Ion microscopes based on the principle of secondary ion mass spectrometry ("SIMS") are known valuable systems used for studying elemental, that is isotopic, distribution in solid samples. The use of these devices is set forth in N. H. Turner et al, Anal. Chem. 56,373R (1984), and G. H. Morrison and G. Slodzian, Anal. Chem. 47,932A (1975). When employed in the non-imaging mode, such instruments have been used extensively in studies involving semiconductors, geologicals, and other nonhydrated specimens. The imaging mode has been recognized as offering great potential for biological studies in that the unique ion optics of the system allow direct microscopic imaging of ions in relation to cell morphology at high resolution. Typical lateral resolution is in the range of 0.5 .mu.m.
In the study of various biological systems, it has been known that ions play a significant role in intracellular regulatory events. With the capability of ion imaging with cell morphology, the ion microscope, therefore, provides a powerful tool used in studies of the role and transport of ions under physiological, pathological and toxicological states in such biological systems. Given the promise of an ion microscope, however, there is a classical problem employed for ion microanalysis of biological samples; the items under test are hydrated. Such samples had to be sectioned and dehydrated before analysis due to the high vacuum requirements for the ion microscope.
Thus, conventional techniques of biological sample preparation such as fixation using chemical fixatives (gluteraldehyde, osmiumtetroxide, and the like) followed by dehydration with organic solvents (ethanol, acetone, and the like) and finally embedding using the plastic resins, suffer to various degrees from artifacts of relocation and the loss of diffusible elements from their native states during such processing.
Cryotechniques emerged as a choice for biological microanalysis. In the absence of a cold stage in the instrument, cryosectioning followed by freeze drying has been used. Freeze drying artifacts, however, cannot be evaluated in this technique of sample preparation. Thus, to study the true distribution and role of ions in biological systems with an ion microscope, it becomes essential to develop a sample stage with cryogenic capability that allows direct analysis of frozen-hydrated biological samples.
Within the prior art, one known technique has been attempted for the Cameca IMS-3f ion microanalyzer. This device is a version of SIMS instrumentation employing a set of ion optics to maintain spatial orientation of secondary ions which leave the sample surface after sputtering by the primary beam. The cold sample stage attempted for use on a IMS-3f ion microanalyzer employed a stage cooled by nitrogen gas which had in turn been cooled by passing in a bath of liquid nitrogen. This cooled gas then passed over a heating element where it was warmed slightly before flowing on the backside of sample holder mount. A thermocouple was used to measure the sample temperature and it was located inside a cold stem where the cold gas flowed passed it before being removed from the system. The sample was held in a standard sample holder having poor thermoconductivity. The thermocouple itself was a large heat sink due to the sample holder bellows in a relatively thick cup to the cold stem. Consequently, the measurements of temperature were at best an inaccurate assessment of the sample. Such is a severe limitation for use with biological samples and consequently this device was not commercially acceptable.